C. difficile is a toxin producing strict anaerobic bacterium which is the causative agent for the diarrheal syndrome termed antibiotic associated pseudomembranous colitis (PMC). Evidence exists which suggests that the pathogenic and cytotoxigenic effects of C. difficile are mediated by two toxins, A and B. The main objective of this proposal is to identify and characterize biologically important regions on these toxins such as antigenic, toxic and binding sites. Toxin gene fragments have been cloned in E. coli in our laboratory: therefore, chromosomal restriction fragments containing full length toxin determinants will be identified, subcloned and mapped. Cytotoxic and binding activity will be monitored for during subcloning experiments, and cloning data will be used to generate a series of gene specific toxin DNA probes. These radiolabeled probes will be hybridized to toxin positive and toxin negative strains of C. difficile, as well as other species of clostridium; thereby, providing information about the chromosomal nature and ubiquity of these toxins. After cloning the toxin genes, DNA sequences will be determined using the dideoxy sequence procedure. Computer analysis of these sequences will be conducted to identify regulatory sites and predict amino acid sequences. Deduction of amino acid sequences will allow for further characterization of the structure of toxins A and B, which remains to be clearly elucidated; as well as allow for the prediction and identification of antigenic sites using hydrophilicity plots, and synthetic oligodeoxynucleotides. ln future studies this data will provide recombinant peptides for immunologic, toxic and binding studies, and may contribute to the development of diagnostic procedures for C. difficile induced PMC.